A number of bacterial metal transporters belong to the “Cluster 9” family of ABC transporters.  The residues in the periplasmic domain thought to be involved in metal binding seem highly conserved and yet the transporters have varying metal specificity.
To solve this seeming paradox and ascertain how metal specificity is exacted, the structure of ZnuA, the periplasmic domain of a zinc transporter from Synechocystis 6803, has been determined to a resolution of 1.9Å.  In previously determined structures of homologous proteins, four residues chelate the bound metal.  From sequence alignments of the “Cluster 9” metal transporters, the fourth residue in this metal binding site, an aspartate, is also present in the appropriate position in the ZnuA sequence.  However, this result is misleading since our structural data indicate that zinc binds via only three histidines and the aspartate is replaced by a large hydrophobic cavity.  We propose that ZnuA binds zinc over manganese by providing only three ligating residues.  ZnuA has a highly charged and mobile loop that protrudes from the protein in the vicinity of the metal binding site.  Similar loops are found in other types of zinc transporters but not manganese transporters.  Therefore, we propose that the function of this domain is to act as a zinc chaperone to facilitate acquisition.  Therefore, while Mn2+ transporters can bind Zn2+ in-vitro they may not be able to acquire it in-vivo without this structure because of the low concentration of free Zn2+.
Divalent metals are essential cofactors in a number of cellular processes.  Bacterial survival and proliferation in the environment as well as within various hosts are critically dependent on the uptake and sequestration of transition metals such as manganese, zinc and iron.  Zinc is an essential element in all organisms and is abundant in the biosphere.  Its Lewis acidity, flexible coordination geometry and rapid ligand exchange properties have led to zinc being the metal of choice in over 300 biological proteins, among them being zinc-finger containing transcription factors and key enzymes such as carbonic anhydrase and superoxide dismutase.  Cells stringently regulate their intracellular zinc levels, since too little zinc inhibits metabolism and high concentrations of zinc is toxic to cellular functions.  To acquire the necessary zinc for metabolism, cells have evolved several types of proteins that are involved in binding and transport of zinc (Claverys 2001).  Zinc transport across the plasma membranes of various bacterial species has been well investigated.  Intracellular levels of zinc in bacteria are maintained within strict limits by the activities of several transport mechanisms involving both the uptake and efflux of zinc (reviewed in (Hantke 2001; Grass, Wong et al. 2002)).        A class of ATP-binding cassette-type (ABC-type) transport system are involved in the uptake of transition metal ions.  This system has several homologues in various gram-positive and gram-negative bacteria that are responsible for the transport of divalent metal cations such as Mn2+ and Zn2+, particularly at low extracellular levels of these metals (Bartsevich and Pakrasi 1996; Claverys 2001; Hantke 2001).  The ABC-type binding proteins from a number of bacteria have been grouped into clusters on the basis of their sequence homologies and the metal ligand identities (Bouige, Laurent et al. 2002).  On this basis, the zinc transporter ZnuA from Escherichia coli (Hantke 2001) and Synechocystis 6803 (Pakrasi 2001), the manganese transporter PsaA from Streptococcus pneumoniae (Dintilhac, Alloing et al. 1997) and the proposed zinc transporter TroA from Treponema pallidum (Deka, Lee et al. 1999; Lee, Deka et al. 1999; Lee, Dorwart et al. 2002) have been placed in a newly defined “Cluster 9” (Claverys 2001).  First identified in E. coli, the znu (zinc uptake) system has been shown to be important for scavenging and transport of Zn2+ (Patzer and Hantke 1998).  Inactivation of genes that encode homologues of znuA has resulted in decreased growth rates and virulence in several pathogenic bacteria (Dintilhac, Alloing et al. 1997; Lewis, Klesney-Tait et al. 1999; Chen and Morse 2001).  In Synechocystis 6803, the znu operon includes the znuA, znuB, and znuC genes that encode the periplasmic Zn-binding protein, the integral membrane protein component, and the cytoplasmic ABC cassette domain, respectively (Shibata et al, manuscript in preparation; (Pakrasi 2001)).      While the overall fold appears to be similar to that of the SBPs from the two transition metal ABC transporters PsaA and TroA, significant differences in the metal binding site offer clues as to the structural mechanism of metal selectivity.  We propose a novel tripartite zinc coordination model based on our structural determination.  A flexible loop, rich in acidic and histidine residues, is found near the entrance to the metal binding site and is absent from the manganese transporters.  Since similar motifs are found in other types of zinc transporters and because the concentration of intracellular free zinc is exceedingly low, we propose that such loops act as zinc chaperones to facilitate the sequestering of zinc around the metal binding site.
	Shown here is a stereo diagram of the electron density map (2Fo-Fc) of the area surrounding the bound metal, contoured at 1.0s.  The zinc ion and the water molecule are represented by purple and red spheres, respectively.  The residues interacting with the bound zinc are represented by a stick model colored by atom type (carbon atoms in yellow, oxygen in red, and nitrogen in blue).
A comparison of sequences of periplasmic solute (metal) binding proteins, SBPs classified as belonging to Cluster 9 of the ABC-type binding proteins.  The names of the SBPs shown are found in the associated bacteria as follows: Syn-ZnuA (Synechocystis 6803), E.Coli-ZnuA (Escherichia coli), Spn-AdcA (Streptococcus pneumoniae), TroA (Treponema pallidum), PsaA (Streptococcus pneumoniae) and Syn-MntC (Synechocystis 6803). Identical residues are highlighted in yellow.  Positions corresponding to the first three ligand binding residues are boxed in black.  Two positions of complete conservation are highlighted in green these correspond to D-313 and E-290 in Syn – ZnuA, respectively.  D-313 is at a conserved residue at positions thought to be the fourth ligand for binding the metal moiety.  E-290 is a conserved position in 95% of all periplasmic metal binding proteins that are grouped in Cluster 9.  Positions known to be involved in secondary shell stabilization of ligand binding residues are boxed or circled in blue.  The secondary shell positions for Syn – ZnuA are indicated above the sequence with a red asterix.

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